Download Inhibition of Acetylcholinesterase in Different Structures of the Rat Brain Following Soman Intoxication Pretreated with Huperzine A

Int. J. Mol. Sci. 2007, 8, 1165-1176
International Journal of
Molecular Sciences
ISSN 1422-0067
© 2007 by MDPI
http://www.mdpi.org/ijms
Full Research Paper
Inhibition of Acetylcholinesterase in Different Structures
of the Rat Brain Following Soman Intoxication
Pretreated with Huperzine A
Jiri Bajgar 1,*, Petr Hajek 2,†, Jana Karasova 1, ‡, Dasa Slizova 2, Otakar Krs 2, Kamil Kuca 1,3,
Daniel Jun 1,3, Josef Fusek 1 and Lukas Capek 4
1 Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Trebesska
1575, 500 01 Hradec Kralove, Czech Republic; ‡E-mail: [email protected]
2 Department of Anatomy, Medical Faculty of Charles University, Hradec Kralove, Czech Republic;
†
E-mail: [email protected]
3 Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec
Kralove, Czech Republic
4 Department of Engineering Mechanics, Faculty of Mechanical Engineering, Technical University of
Liberec, Liberec, Czech Republic
* Author to whom correspondence should be addressed; E-mail: [email protected];
Tel: +420 973 251 507; Fax: +420 495 518 094;
Received: 15 October 2007; in revised form: 14 November 2007 / Accepted: 15 November 2007 /
Published: 26 November 2007
Abstract: Acetylcholinesterase (AChE) activities in different brain parts were determined
quantitatively in rats treated with huperzine A, soman, and huperzine A followed by
soman, using histochemical and biochemical methods. Following soman intoxication (1.2
x LD50, i.m.), AChE activity was decreased to 30-80% of control values depending on the
brain structure. The most sensitive area was the frontal cortex and the most relatively
resistant was ncl. ruber. Huperzine A treatment only caused a change in AChE activity
varying from 70 to 100 % of control values. In rats pretreated with huperzine A and
intoxicated with soman, AChE activity was significantly higher than that observed after
soman. In these animals, survival of rats pretreated with huperzine was observed while
the mortality of unpretreated animals was near to 80 %. The results suggest that huperzine
A is good candidate for further study for clinical use as a prophylactic drug against nerve
agent poisoning.
Int. J. Mol. Sci. 2007, 8
1166
Keywords: acetylcholinesterase, rat brain parts, soman, Huperzine A, pretreatment
1. Introduction
Nerve agents belong to the most toxic organophosphorus cholinesterase inhibitors. They can be
used as chemical warfare agents and, they can be (and have been) misused by terrorists. The
countermeasures against their effect (i.e. prophylaxis and treatment) are of high importance [1-4].
Protection against the inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) using reversible
cholinesterase inhibitors is the most common approach to pharmacological prophylaxis. The
administration of present antidotes or scavengers such as cholinesterase preparations to prevent the
effects of OP is also possible [1,5,6]. As for as protection against cholinesterase inhibition,
pyridostigmine seems to be a most common prophylactic drug. However, its low central effects and
relative high toxicity are disadvantages of its use. Therefore a search for new prophylactics was
performed.
Huperzine A is reversible and selective AChE inhibitor compared to BuChE and is a more potent
inhibitor of AChE than pyridostigmine. The review of Jiang et al. [7] represents a comprehensive
documentation of the progress in the studies on huperzine A covering the period 1999-2002. It appears
to be a selective inhibitor of red cell AchE, while pyridostigmine or physostigmine additionally inhibit
plasmatic butyrylcholinesterase (BuChE, EC 3.1.1.8) in guinea-pigs [8,9]. Since pyridostigmine does
not penetrate into the brain, it does not afford protection against seizures and subsequent
neuropathology induced by an OP agent such as soman. Besides, huperzine has also been successfully
tested for pretreatment of OP poisoning [10,11].
Huperzine A was tested for its prophylactic potential [12-17] and by means of a dynamically
working in vitro model using AChE from immobilized human erythrocytes. The model predicts that
inhibitors with a faster dissociation constant such as huperzine may be superior in case of very
effective poisons such as soman [18]. However, there is a lack of information dealing with selective
effect of huperzine especially on the AChE in the brain structures. It is known that the brain is wellorganized and complex organ containing different levels of neuromediators and relevant enzymes in
various structures of the brain. AChE activity varies a minimum 14-fold between striatum and
cerebellum [19]. In prophylactic studies, AChE activity was determined in the whole brain
homogenate. Some our results showed differential AChE inhibition in various brain regions, ranging
from 15 to 86% following intoxication with soman [20]. Thus, the activity determined in the whole
brain homogenate can be considered as a “mean” of the activities in different brain structures. It is
difficult to correlate biochemical results which are not defined structurally as those in histochemical
studies. However, this approach (comparison of these results) was used [20] and showed that it is
possible and it would be interesting approach allowing more detailed explanation of action of nerve
agents as well as their therapeutic influencing. Therefore (on the basis of above mentioned studies),
prophylactic effects of huperzine A against soman using determination of AChE activity
(histochemical and biochemical) in selected rat brain structures was studied.
Int. J. Mol. Sci. 2007, 8
1167
2. Results and Discussion
2.1 Mortality
Following soman treatment, five from six animals died within 30 min after soman poisoning.
Typical symptoms of poisoning – salivation, disturbed ventilation and convulsions – were observed in
all animals in this group. After administration of huperzine A, fine tremor was observed occuring 1030 min post administration, as well as in a group of animals pretreated with huperzine A and
intoxicated with soman. All animals in these two groups showed practically unchanged ventilation and
all animals survived.
2.2 Biochemical
The data on AChE activity in the brain areas are presented in Table 1. AChE activity varied from
high (F.ret, Th, Hipp) to low (FC, NR) values.
Table 1. Histochemical (H) and biochemical (B) results of AChE activities following
Huperzine A and soman treatment.
Group
CONTROL
Area
FC
Hipp
HTh
H
B
SOMAN
HUPERZINE
H
B
H
B
HUPERZINE+SOMAN
H
B
a
181.1
253.0±13.3
53.4
70.1±10.5
125.6
190.2±12.1
83.8
121.4±12.4
%
100
100
29.V
27.7c
69.3
75.2a,d
46.3
48.0 b,c,d
a
221.45
301.6±15.3
89.5
135.5±14.7
165.9
241.3±14.6
112.5
158.2±13.8
50.8
52.5 b,d
154.7
209.6±14.3
76.0
80.0 b,c,d
%
100
100
40.4
44.9
74.9
a
203.4
262.0±14.3
66.8
92.7±13.8
184.2
c
%
100
100
32.8
35.4
a
187.9
251.3±15.3
141.05
198.3±14.3
c
80.0
a,d
249.1±14.2
d
90.6
95.0
198.35
261.4±13.9
176.4
246.0±14.1
105.6
104.0
93.9
97.9 c
NR
%
100
100
75.1
78.9
a
226.9
385.0±13.3
63.7
101.5±12.3
176.5
269.5±12.9
147.4
261.8±13.5
F.ret
%
100
100
28.I
26.4c
77.8
70.0a
65.0
68.0 b,c
a
192.7
251.2±12.3
57.7
89.6±10.8
152.3
213.5±13.0
154.7
205.8±13.1
80.3
81.9 b,c
172.6
253.2±14.2
77.1
77.8b,c,d
DSep
Th
c
%
100
100
29.IX
35.7
a
223.9
325.3±14.0
74.1
114.8±13.7
%
100
100
33.1
35.3
c
a
79.0
85.0
214.1
322.0±13.5
95.6
99.0
d
For biochemical examinations, the results are means with their standard errors (n=6) (AChE activity in
µmol/60 min/kg) and percent activity expression (%).
For histochemical examinations, the results are means (AChE activity in pixel density, n=3) and percent
activity expression (%).
BIOCHEMICAL RESULTS: All control values are significantly different from group soman (all parts)
(p<0.05). The letters indicate significant difference for other experimental groups: (NR – nucleus ruber
from amygdala, FC – frontal cortex, DSep – dorsal septum, HTh – hypothalamus, Hipp – hippocampus, Th
– thalamus, F.ret – pontomedullar area, mostly reticular formation containing nucleus gigantocellularis and
others).
Int. J. Mol. Sci. 2007, 8
1168
Administration of soman caused strong inhibition of AChE in all areas studied. Percentual
inhibition was highest in F.ret and FC, which had about 30 % of the control activity. For huperzine
treatment, the highest AChE inhibition was observed in F.ret and FC areas (about 70-75 % of controls).
Lower inhibition of AChE observed in the rest brain structures and in NR, AChE inhibition was not
detected.
Following pretreatment with huperzine and soman intoxication (HUPERZINE+SOMAN group),
AChE activity in all brain parts examined was lower in comparison with huperzine treatment only
(HUPERZINE group). However, the activities were higher in all brain parts compared with soman
intoxication only (SOMAN group), and they were statistically significant for NR, F.ret, and DSep,
respectively.
2.3 Histochemical
The histochemical results showed a similar picture to that of the biochemical examination (Table
1). The area most sensitive to soman was FC and Dsep (about 30 % of control values), and the most
resistant was NR (75 %). The other soman treated brain regions had about 30-40 % of the control
values. Histochemical inhibition values following administration of huperzine only matched most
closely in the FC. The structures most resistant to huperzine seems to be NR. Histochemical pictures of
three areas, the relatively sensitive FC, and F.ret and Hipp are shown in Figures 1-3. The quantitative
evaluation following intoxication with soman pretreated with huperzine is also shown also in Figures
1-3, where it can be seen that AChE inhibition in all parts has had relatively same tendency as observed
for biochemical examinations. According to biochemical results, AChE inhibitions following
huperzine administration (HUPERZINE group) and soman intoxication pretreated with huperzine
(HUPERZINE+SOMAN group) are very similar. However, after quantitative histochemical evaluation,
the activities are shifted to lower densities and frequency of pixels is different.
Figure 1. Top: Microphotography of 20 µm sections of the rat brain (reticular formation,
ncl. gigantocellularis, position is marked by arrow) following huperzine, soman and
huperzine followed by soman administration. 1 - control, 2 - Huperzine A, 3 – soman, 4
– Huperzine A + soman. Magnification: 40x, Staining: AChE [22].
1
2
3
4
Int. J. Mol. Sci. 2007, 8
1169
frequency of pixels
Bottom: Quantitative evaluation of histochemical data: Density curves of microphotographs.
25000
control
Huperzine A
20000
15000
10000
pixel density
5000
0
14 24 34 44 54 64 74 84 94104114124134144154164174184194204214224
Figure 2. Top: Microphotography of 20 µm sections of the rat brain (hippocampus)
following huperzine, soman and huperzine followed by soman administration. 5 - control,
6 - Huperzine A, 7 – soman, 8 – Huperzine A + soman. Magnification: 40x, Staining:
AChE [22].
5
6
7
8
Bottom: Quantitative evaluation of histochemical data: Density curves of microphotographs
25000
hippocampus
control
Huperzine A
20000
frequency of pixels
soman
Huperzine A + soman
15000
10000
5000
0
9
19
29
39
49
59
69
79
89
99
109
119
pixel density
129
139
149
159
169
179
189
199
209
219
Int. J. Mol. Sci. 2007, 8
1170
Figure 3. Top: Microphotography of 20 µm sections of the rat brain (frontal cortex)
following huperzine, soman and huperzine followed by soman administration. 9 –
control, 10 - Huperzine A, 11 – soman, 12 – Huperzine A + soman. Magnification: 40x,
Staining: AChE [22].
9
10
11
12
Bottom: Quantitative evaluation of histochemical data: Density curves of microphotographs.
140000
frontal cortex
120000
control
Huperzine A
frequency of pixels
100000
soman
Huperzine A + soman
80000
60000
40000
20000
0
24
34
44
54
64
74
84
94
104
114
124
134
144
154
164
174
184
194
204
214
224
234
244
pixel density
Correlation between results obtained using histochemical and biochemical methods
The comparison of percentage of residual AChE activity after administration of soman, huperzine
and huperzine followed by soman in 7 brain regions (Figure 4) shows a good correlation between
results obtained by histochemical and biochemical methods of AChE detection.
Discussion
Huperzine A is considered and studied with respect to prophylaxis in vitro and in vivo [11,14,16].
In general, reversible inhibitors including huperzine protect organism from the acute toxicity of lethal
doses of the nerve agents such as sarin or soman [1,5,10,11,13] as it was also demonstrated in our
experiments.
At the light of its particular inhibitory affects on the brain AChE activity, huperzine, compared to
the other drugs, seems to be the optimal candidate to be used as pretreatment against OP poisoning
[8,9] as demonstrated in guinea-pigs. In preventing the lethality of nerve agents, huperzine was
effective compared with other reversible cholinesterase inhibitors (Tacrine, donepezil, rivastigmine,
Int. J. Mol. Sci. 2007, 8
1171
etc.) [17]. The protective effects against soman toxicity of huperzine were demonstrated also in
primates [10].
Figure 4. Comparison of results obtained by histochemical and biochemical methods
(percentual values) following administration of soman, huperzine and soman pretreated
with huperzine.
biochemical
(% of control values)
Fig. 4: Comparison of results obtained by
biochemical and histochemical methods
(percentual values)
150
100
y = 1.022x + 0.3738
2
R = 0.9693
50
0
0
50
100
150
histochemical (% of control values)
The results of Grunwald et al. [12] suggest that the protection of mice from soman poisoning by
huperzine was achieved by temporary sequestering the active site region of the physiologically
important AChE. In this connection, it is of interest that the main cholinergic pathways in the brain
cholinergic system are represented i.a by septal nuclei, thalamus, cortex and hippocampus [26]. Some
of these structures were studied in our paper and their influencing either by soman or huperzine was
different. The not-uniform AChE inhibition in the different brain structures following untreated
intoxication with nerve agents in rats and guinea-pigs [27,28] was demonstrated. This non-uniform
inhibition suggests that some brain structures more sensitive to huperzine or OP than others can be
functionally important for toxic/prophylactic action. The pontomedullar area seems to be the important
structure because of its role in the respiratory control through the regulatory centers for respiration [29]
in this brain part. Respiration is under cholinergic innervation [29,30]. The depression of central
respiratory control centers in the pontomedullar area is considered as a primary event leading to death
[31-33]. The highest AChE inhibition in this structure either by soman or huperzine in vivo supports a
hypothesis that AChE inhibition here could be a cause of an interuption of cholinergic pathways
controlling ventilation function. This not-uniformity can be explained by fine differences of huperzine
action not only on the enzyme (AChE) activity but also by its different action on a structural level.
Nucleus gigantocellularis contained in pontomedullar area and AChE here could be a key target for a
survival or death of animals intoxicated with soman. On the other hand, AChE activity in NR was
relatively unchanged in groups pretreated with huperzine only and huperzine followed by soman
Int. J. Mol. Sci. 2007, 8
1172
intoxication. This resistancy supports an idea about different penetration of drugs/poisons into various
brain areas.
These results suggest that huperzine is an important drug employed not only in the treatment of
Alzheimer disease [26] but it is good candidate for further study for clinical use as prophylaxis against
nerve agents. Its generally known effect – protection of AChE against its inhibition by soman – would
have different importance for various brain structures. Our results underline the importance of
pontomedullar brain area in this connection.
3. Experimental Section
3.1 Animals
Female Wistar rats (VELAZ Prague), weighing 200-220 g, were used in this study. The animals
were divided into groups of three (histochemical) and six (biochemical) animals each. Rats were
housed in the Central Vivarium of the Faculty of Military Health Sciences under veterinary control. All
the experiments were performed under permission and supervision of the Ethics Committee of the
Faculty of Military Health Sciences, Hradec Kralove (permission No 153/06) according to § 17 of the
Czech law No 207/2004, permission of responsible person 0001/94 – M 699.
3.2 Chemicals
Soman was obtained from the Military Technical Institute of Protection (Brno, Czech Republic). It
was of minimally 99% purity and stored in glass ampullas (1 mL). The solutions of the agents for
experiments were prepared before use. Huperzine A was obtained from Sigma Aldrich Gmbh (Division
of Czech Republic) as well as other chemicals of analytical purity.
3.3 Intoxication and pretreatment
CONTROL GROUP: The animals were injected with saline i.p. and 30 min later, they were injected
once again with saline i.m. (0.1 mL/kg). The decapitation and brain removal (sampling) was realized
30 min after the last saline injection.
SOMAN GROUP: The animals were injected with saline i.p. and 30 min later, soman was
administered i.m. in a dose of 1.2 x LD50, i.e. 84.0 µg/kg; 30 min after the injection/at the time of
death, the animals were decapitated, the brain was removed and used for histochemical or biochemical
examinations.
HUPERZINE GROUP: The animals were injected i.p. with Huperzine A (500 µg/kg) and 30 min
later, they were injected with saline i.m. The dose of Huperzine A as well as the route of administration
was used on the base of previously published results [12]. The sampling was performed 30 min after
the injection of saline.
Int. J. Mol. Sci. 2007, 8
1173
HUPERZINE+SOMAN GROUP: the animals were pretreated with Huperzine as it is described in
the previous group. 30 min later, the animals were intoxicated with soman and decapitated 30 min after
the soman injection.
3.4 Histochemical determination of AChE
Removed brains were rapidly frozen and cut into a series of 20 µm sections in a cryostat. For
neuroanatomical mapping according to the rat brain atlas [21], AChE detection [22] was used. The
Karnovski-Roots method is based on hydrolysis of artificial substrate acetylthiocholine (the same as in
biochemical examination) and detection of the released reaction product (thiocholine). For digital
microphotography, Olympus BX51 light microscope equipped with CCD was used.
Quantitative evaluation was made using software 3D Doctor [23,24]. The picture was transposed to
a gray scale with density distribution (expressed in pixels) ranging from 0 to 255. Lower pixel number
indicates high activity, higher number shows inhibition. Because the density is of linear scale, the
difference (255 - determined density) gives us information on the AChE activity. This pixel density
was compared in control and intoxicated animals for each structure examined in absolute and relative
(%) values.
We selected the following sections and groups of nuclei to compare quantitative histochemistry
with biochemical determination of AChE: ncl. ruber (NR) – section at the level 5,8 dorsally from
bregma; frontal cortex (FC) – the level of 2.2 mm rostrally to bregma, containing areas F1, F2, F3;
dorsal septum (DS) – the level of bregma, including lateral septal ncl – dorsal and intermediate parts;
hypothalamus (HTh), 2.8 mm dorsally to bregma, including ventromedial and dorsomedial
hypothalamic nuclei, perifornical, arcuate, periventricular and supraoptic nuclei, tuber cinereum, dorsal
and lateral hypothalamic areas; hippocampus (Hipp), 3.5 mm dorsally to bregma; medial thalamus (Th)
2,8mm dorsally from bregma including mediodorsal nuclei, intermediodorsal, paraventricular,
paracentral, central medial and centrolateral nuclei; ponto-medullar area (F.ret, section at the level of
13 mm dorsally to bregma, we evaluated only ncl. gigantocellularis as representative by AChE activity,
its size, and function. The distance from bregma and terminology of nuclei are quoted from Paxinos
and Watson [21].
3.5 Biochemical determination of AChE
The brain parts (NR, FC, DS, HTh, Hipp, Th, F.ret) were prepared and frozen. After thawing, tissue
was homogenized (1:10, distilled water, Ultra Turrax homogenizer) and homogenates were used for
enzymatic analysis. AChE activity was determined using the method of Ellman et al. [25].
Acetylthiocholine was used as substrate (Tris-HCl buffer pH 7.6). UVIKON 752 spectrophotometer
was used for determination of absorbancy at 412 nm. The activity was expressed as µmol of substrate
hydrolyzed/(60 min.kg) wet weight tissue or as % of control values.
3.6 Statistical evaluation
Int. J. Mol. Sci. 2007, 8
1174
Enzyme activities were expressed as a mean ±SD or % of control values (means only) and statistical
differences were tested by t-test. Transformation of the curves and their equations and correlation
coefficients were evaluated by the least square method using relevant PC programmes.
Acknowledgements
The authors are indebted to Mrs J. Ellingerova and J. Uhlirova for skilful technical assistance.
Financial support of the Ministry of Defence (grant No OPUOFVZ 200603) and Grant Agency of
Charles University (grant No GA UK 82/2005) is gratefully acknowledged.
References and Notes
1.
Bajgar, J. Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis
and treatment. Adv. Clin. Chem. 2004, 38, 151-216.
2. Eyer, P. The role of oximes in the menagement of organophosphorus pesticides poisoning.
Toxicol. Rev. 2003, 22, 165-190.
3. Rotenberg, J.S.; Newmark, J. Nerve attacks on children: diagnosis and management. Pediatrics
2003, 112, 648-658.
4. Wiener, S.W.; Hoffman, R.S. Nerve agents: a comprehensive review. J. Int. Care. Med. 2004, 19,
22-37.
5. Bajgar J. Prophylaxis against organophosphorus poisoning. J. Med. Chem. Def. 2003, 1, 1-15.
6. Bajgar, J.; Fusek, J.; Sevelova, L.; Kassa, J. Complex prophylaxis against nerve agent
intoxication. In: The Eighth International Symposium on Protection against Chemical and
Biological Warfare Agents, Goteburg, Sweden, 2-6 June 2004, Abstracts; 2004; p. 51.
7. Jiang, H.L.; Luo, X.M.; Bai, D.L. Progress in clinical, pharmacological, chemical and structural
biological studies of huperzine A: a drug of traditional Chinese medicine origin for the tratment of
Alzheimer´s disease. Curr. Med. Chem. 2003, 10, 2231-2252.
8. Lallement, G.; Foquin, A.; Dorandeu, F.; Baubichon, D.; Aubriot, S.; Carpentier, P. Subchronic
administration of various pretreatments of nerve agent poisoning. I. Protection of blood and
central cholinesterases, innocuousness towards blood-brain barrier permeability. Drug Chem.
Toxicol. 2001, 24, 151-164.
9. Lallement, G.; Foquin, A.; Dorandeu, F.; Baubichon, D.; Carpentier, P. Subchronic administration
of various pretreatments of nerve agent poisoning. II. Compared efficacy against soman toxicity.
Drug Chem. Toxicol. 2001, 24, 165-180.
10. Lallement, G.; Demoncheaux, J.P.; Foquin, A.; Baubichon, D.; Galonnier, M.; Clarencon, D.;
Dorandeu, F. Subchronic administration of pyridostigmine or huperzine to primates: compared
efficacy against soman toxicity. Drug Chem. Toxicol. 2002, 25, 309-320.
11. Lallement, G.; Taille, V.; Baubichon, D.; Carpentier, P.; Collombet, J.M.; Filliat, P.; Foquin, A.;
Four, E.; Masqueliez, C.; Testylier, G.; Tondulli, L.; Dorandeu, F. Review of the value of
huperzine as pretreatment of organophosphate poisoning. Neurotoxicology 2002, 23, 1-5.
Int. J. Mol. Sci. 2007, 8
1175
12. Grunwald, J.; Raveh, L.; Doctor, B.P.; Ashani, Y. Huperzine-A as a pretreatment candidate drug
against nerve agent toxicity. Life Sci. 1994, 54, 991-997.
13. Albuquerque, E.X.; Pereira, E.F.R.; Aracava, Y.; Fawcett, W.P.; Oliveira, M.; Randall, W.R.;
Hamilton, T.A.; Kan, R.K.; Romano, J.A.; Adler, M. Effective countermeasures against poisoning
by organophosphorus insecticides and nerve agents. Proc. Nat. Acad. Sci. USA 2006, 103, 1322013225.
14. Ashani, Z.; Peggins, J.O.; Doctor, B.P. Mechanism of inhibition of cholinesterase by Huperzine A.
Biochem. Biophys. Res. Commun. 1992, 184, 719-726.
15. Gordon, R.K.; Haigh, J.R.; Garcia, G.E.; Feaster, S.R.; Riel, M.A.; Lenz, D.E.; Aisen, P.S.;
Doctor, B.P. Oral administration of pyridostigmine bromide and huperzine A protects human
whole blood cholinesterases from ex in vivo exposure to soman. Chem-Biol. Interact. 2005, 157,
239-246.
16. Patocka, J.; Kassa, J. Huperzine A – prospective prophylactic antidote against organophosphate
warfare agent poisoning. ASA Newslett. 1999, 99-2, 16-19.
17. Wang, R.; Yan, H.; Tang, X.C. Progress in studies of huperzine A, a natural cholinesterase
inhibitor from Chinese herbal medicine. Acta Pharmacol. Sin. 2006, 27, 1-26.
18. Eckert, S.; Eyer, P.; Muckter, H.; Worek, F. Kinetic analysis of the protection afforded by
reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic
organophosphorus compounds. Biochem. Pharmacol. 2006, 72, 344-357.
19. Gupta, R.C. Brain regional heterogeneity and toxicological mechanisms of organophosphates and
carbamates. Toxicol. Mech. Methods 2004, 14, 103-143.
20. Bajgar, J.; Hajek, P.; Slizova, D.; Krs, O.; Fusek, J.; Kuca, K.; Jun, D.; Bartosova, L.; Blaha, V.
Changes of acetylcholinesterase activity in different brain areas following intoxication with nerve
agents: biochemical and histochemical study. Chem-Biol. Interact. 2007, 165, 14-21.
21. Paxinos, G.; Watson, C. The rat brain in stereotactic coordinates. Academic Press: New York,
1987.
22. Lojda, Z.; Gossrau, R.R.; Schiebler, T.H. Enzyme Histochemistry – A Laboratory Manual.
Springer: New York, 1979.
23. Benali, A.; Leefken, I.; Eysel, U.; Weile, E. A computerized image analysis system for
quantitative analysis of cells in histological brain sections. J. Neurosci. Meth. 2003, 125, 33-43.
24. Hammond, P.I.; Jelacic, T.; Padilla, S.; Brimijoin, S. Quantitative, video-based histochemistry to
measure regional effects of anticholinesterase pesticides in rat brain. Anal. Biochem. 1996, 241,
82-92.
25. Ellman, G.L.; Courtney, D.K.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88-95.
26. Giacobini, E.; Pepeu G. Cholinesterases in human brain: the effect of cholinesterase inhibitors on
Alzheimer´s disease and related disorders. In The Brain Cholinergic System in Health and
Disease; Informa Healthcare: Abingdon, UK, 2006; pp. 235-264.
27. Gupta, R.C.; Patterson, G.T.; Dettbarn, W.D. Biochemical and histochemical alterations following
acute soman intoxication in the rat. Toxicol. Appl. Pharmacol. 1987, 87, 393-402.
Int. J. Mol. Sci. 2007, 8
1176
28. Shih, T.-M.; Kan, R.K.; McDonough, J.H. In vivo cholinesterase inhibitory specificity of
organophosphorus nerve agents. Chem-Biol. Interact. 2005, 157, 158, 293-303.
29. Pope, C.N. Central nervous system effects and neurotoxicity. In Toxicology of Organophosphate
and Carbamate Compounds; Elsevier-Academic Press: Amsterdam, 2006; pp. 271-291.
30. Sungur, M.; Guven, M. Intensive care management of organophosphate insecticide poisoning.
Crit. Care 2001, 5, 211-215.
31. Goswany, R.; Chaudhuri, A.; Mahashur, A.A. Study of respiratory failure in organophosphate and
carbamate poisoning. Heart Lung 1994, 23, 466-472.
32. Stewart, W.C.; Anderson, E.A. Effect of a cholinesterase inhibitor when injected into the medulla
of the rabbit. J. Pharmacol. Exp. Ther. 1968, 162, 309-318.
33. Bird, S.B.; Gaspari, R.J.; Dickson, E.W. Early death due to severe organophosphate poisoning is a
centrally mediated process. Acad. Emerg. Med. 2003, 10, 295-298.
© 2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.